Methods for identifying compounds for the treatment of type 1 diabetes

ABSTRACT

The present invention provides methods of identifying candidate compounds for the treatment of type I diabetes comprising contacting pancreatic β cells with an amount of apolipoprotein CIII (“apoCIII”) effective to increase intracellular calcium concentration, in the presence of one or more test compounds, and identifying those test compounds that inhibit an apoCIII-induced increase in intracellular calcium concentration in the pancreatic β cells. The present invention also provides methods for treating patients with type I diabetes comprising administering to the patient an amount effective of an inhibitor of apoCIII to reduce apoCIII-induced increase in intracellular calcium concentration in pancreatic β cells.

CROSS REFERENCE

This application is a divisional of U.S. application Ser. No. 10/834,525filed Apr. 29, 2004, and claims priority to U.S. Provisional PatentApplication 60/466,517 filed Apr. 29, 2003.

BACKGROUND OF THE INVENTION

Voltage-gated L-type Ca²⁺-channels have an important physiological rolein pancreatic β-cell (“β-cell”) signal-transduction (1). These channelsconstitute an essential link between transient changes in membranepotential and insulin release from β-cells. Changes in cytoplasmic freeCa²⁺ concentration ([Ca²⁺]_(i)) in the β-cell are associated with theactivation of a spectrum of intracellular signals and are strictlyregulated, as prolonged high [Ca²⁺]_(i) is harmful to the cells.

In type 1 diabetes (T1D), there is a specific destruction of the insulinsecreting pancreatic β-cell. Sera from newly diagnosed type 1 diabetic(T1D) patients have been shown to increase the activity of voltage-gatedL-type Ca²⁺-channels in β-cells resulting in increased [Ca²⁺]_(i) upondepolarization and β-cell apoptosis, effects that can be prevented byCa²⁺-channel blockers (2). However, it has not been determined whatfactor in T1D serum is responsible for the changes in [Ca²⁺]_(i).

We now demonstrate that apolipoprotein CIII (apoCIII) is increased inserum from T1D patients and that this serum factor both inducesincreased cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_(i)) and β-celldeath.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods of identifyingcandidate compounds for the treatment of type I diabetes comprisingcontacting pancreatic β cells with an amount of apolipoprotein CIII(“apoCIII”) effective to increase intracellular calcium concentration,in the presence of one or more test compounds, and identifying thosetest compounds that inhibit an apoCIII-induced increase in intracellularcalcium concentration in the pancreatic β cells. In another aspect, thepresent invention provides methods for treating patients with type Idiabetes comprising administering to the patient an amount effective ofan inhibitor of apoCIII to reduce apoCIII-induced increase inintracellular calcium concentration in pancreatic β cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Changes in [Ca²⁺]_(i) in pancreatic β-cells from mice exposed toT1D or control sera. Five out of seven T1D sera induced an enhancedincrease in [Ca²⁺]_(i), when the cells were depolarized with highconcentrations of K⁺ to open the voltage-gated Ca²⁺-channel, compared tocells that had been exposed to normal serum (n=29, 28, 32, 47, 21, 27,31 and 18, respectively), *** P<0.001, ** P<0.01 and * P<0.05 versuscontrol.

FIG. 2 Stepwise separation and identification of the active fraction inT1D serum. A, After the first RP-HPLC separation the fraction marked 3was found to give a higher increase in [Ca²⁺]_(i). B, Fraction 3 (FIG.2A) was rerun on RP-HPLC under identical conditions. The fractions wereagain tested for [Ca²⁺]_(i) stimulating activity (FIG. 2C), and onepositive fraction (No. 2) was identified. D, The active fraction (FIG.2B) was re-chromatographed. The fraction, inducing a higher increase in[Ca²⁺]_(i) when β-cells were depolarized with high concentrations of K⁺,is marked with a bar. C, Pancreatic β-cells incubated with fourfractions from RP-HPLC of diabetic sera from FIG. 2B (n=6, 11, 12, 11and 10, respectively), *** P<0.001 versus control. E, The activefraction from FIG. 2C was analyzed by electrospray mass spectrometry.

FIG. 3 Amounts of apoCIII in T1D serum and effects on [Ca²⁺]_(i) andcell death. A, Relative levels of apoCIII₁₊₂ in T1D and control serum,given as area under the curve (AUC), **P<0.01 (n=5). B, Pancreaticβ-cells were incubated with apoCIII or apoCIII+antibodies against humanapoCIII (n=63, 35 and 33), ***P<0.001 versus control. C, β-cells wereincubated with a control or a T1D serum and T1D serum+anti-apoCIII(n=18, 17 and 20), ***P<0.001 versus T1D serum. D, Mouse β-cells wereincubated with apoCIII₀, apoCIII₁, apoCIII₂ or the vehicle (control)(n=54, 40, 48, 37), *** P<0.001 versus control. E, The insulin secretingcell line RINm5F was exposed to control and T1D sera and T1D serum withthe addition of two concentrations of anti-apoCIII and finally controlserum with apoCIII (n=5), * P<0.05 and **P<0.01, versus control.

FIG. 4 Interaction of apoCIII with the voltage-gated L-type Ca²⁺channel. A, Summary graph of current density-voltage relationships.ApoCIII-treated cells (filled circles, n=56) and control cells (opencircles, n=55) were depolarized to potentials between −60 and 50 mV, in10 mV increments, from a holding potential of −70 mV, *P<0.05. B, Samplewhole-cell Ca²⁺ current traces from a control cell (cell capacitance:4.3 pF) and a cell incubated with apoCIII (cell capacitance: 4.2 pF).Cells were depolarized by a set of voltage pulses (100 ms, 0.5 Hz)between −60 and 50 mV, in 10 mV increments, from a holding potential of−70 mV.

DETAILED DESCRIPTION OF THE INVENTION

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog(Ambion, Austin, Tex.).

In one aspect, the present invention provides methods of identifyingcandidate compounds for the treatment of type I diabetes comprisingcontacting pancreatic β cells with an amount effective of apolipoproteinCIII (“apoCIII”) effective to increase intracellular calciumconcentration in the presence of one or more test compounds, andidentifying those test compounds that inhibit apoCIII-induced increasein intracellular calcium concentration in the pancreatic β cells.

As used herein, “apoCIII” refers to a protein comprising the amino acidsequence shown in SEQ ID NO:2 (Human) (NCBI accession number CAA25233),SEQ ID NO:4 (Rat) (NCBI accession number AA40746), or SEQ ID NO:6(Macaque) (NCBI accession number CAA48419), or functional equivalentsthereof.

The apoCIII may be substantially purified apoCIII, available, forexample, from Sigma Chemical Company (St. Louis, Mo.), wherein“substantially purified” means that it is removed from its normal invivo cellular environment. Alternatively, the apoCIII may be present ina mixture, such as blood serum from type I diabetic or partially orfully purified therefrom using standard techniques, such as thosedescribed below. In a preferred embodiment, substantially purifiedapoCIII is used.

As discussed below, there are three known isoforms of human apoCIII thathave the same amino acid sequence, but which differ in theirglycosylation pattern. Thus, in a preferred embodiment, glycosylatedapoCIII is used, wherein the glycosylation is preferably sialylation. Inan especially preferred embodiments, mono-sialylated or di-sialylatedapoCIII is used. Such glycosylated forms may be purchased, for example,from Sigma Chemical Company, or may be partially or fully purified usingstandard techniques, such as those described below.

As used herein, “pancreatic β cells” are any population of cells thatcontains pancreatic β islet cells. The cells can be obtained from anymammalian species, or may be present within the mammalian species whenthe assays are conducted in vivo. Such pancreatic β islet cellpopulations include the pancreas, isolated pancreatic islets ofLangerhans (“pancreatic islets”), isolated pancreatic β islet cells, andinsulin secreting cell lines. Methods for pancreatic isolation are wellknown in the art, and methods for isolating pancreatic islets, can befound, for example, in Cejvan et al., Diabetes 52:1176-1181 (2003);Zambre et al., Biochem. Pharmacol. 57:1159-1164 (1999), and Fagan etal., Surgery 124:254-259 (1998), and references cited therein. Insulinsecreting cell lines are available from the American Tissue CultureCollection (“ATCC”) (Rockville, Md.). In a further embodiment wherepancreatic β cells are used, they are obtained from ob/ob mice, whichcontain more than 95% β cells in their islets, and are commerciallyavailable.

As used herein, “intracellular calcium concentration” refers tocytoplasmic free Ca²⁺ concentration ([Ca²⁺]_(i)) in the pancreaticβ-cell. Such concentrations can be measured by any method known in theart, such as the use of fluorescent calcium indicators, as disclosedherein.

As used herein, “increase intracellular calcium concentration” refers toincreasing the concentration during the course of the assay above thatseen in the absence of test compounds. The method does not require aspecific amount of increase in intracellular calcium concentration overbaseline, so long as the compound(s) promotes an increase inintracellular calcium concentration above that seen in the absence oftest compounds. In a preferred embodiment, the increase is astatistically significant increase as measured by standard statisticalmeasurements.

The contacting of the pancreatic β cells with the apoCIII may occurbefore, after, or simultaneously with contacting the cells with one ormore test compounds. The contacting can be in vitro, in vivo, or exvivo.

The present invention further provides compounds identified by the abovescreening methods, and their use for treating subjects with type Idiabetes.

In another embodiment, the methods further comprise synthesizing thetest compounds that inhibit apoCIII-induced increase in intracellularcalcium concentration in the pancreatic β cells.

When the test compounds comprise polypeptide sequences, suchpolypeptides may be chemically synthesized or recombinantly expressed.Recombinant expression can be accomplished using standard methods in theart, as disclosed above. Such expression vectors can comprise bacterialor viral expression vectors, and such host cells can be prokaryotic oreukaryotic. Synthetic polypeptides, prepared using the well-knowntechniques of solid phase, liquid phase, or peptide condensationtechniques, or any combination thereof, can include natural andunnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resinwith standard deprotecting, neutralization, coupling and wash protocols,or standard base-labile Nα-amino protected 9-fluorenylmethoxycarbonyl(Fmoc) amino acids. Both Fmoc and Boc Nα-amino protected amino acids canbe obtained from Sigma, Cambridge Research Biochemical, or otherchemical companies familiar to those skilled in the art. In addition,the polypeptides can be synthesized with other Nα-protecting groups thatare familiar to those skilled in this art. Solid phase peptide synthesismay be accomplished by techniques familiar to those in the art andprovided, such as by using automated synthesizers.

When the test compounds comprise antibodies, such antibodies can bepolyclonal or monoclonal. The antibodies can be humanized, fully human,or murine forms of the antibodies. Such antibodies can be made bywell-known methods, such as described in Harlow and Lane, Antibodies; ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1988). In one example, preimmune serum is collected prior to thefirst immunization with, for example, apoCIII. A substantially purifiedapoCIII, or antigenic fragments thereof, together with an appropriateadjuvant, are injected into an animal in an amount and at intervalssufficient to elicit an immune response. Animals are bled at regularintervals, preferably weekly, to determine antibody titer. The animalsmay or may not receive booster injections following the initialimmunization. At about 7 days after each booster immunization, or aboutweekly after a single immunization, the animals are bled, the serumcollected, and aliquots are stored at about −20° C. Polyclonalantibodies against apoCIII can then be purified directly by passingserum collected from the animal through a column to whichnon-antigen-related proteins prepared from the same expression systemwithout apoCIII bound.

Monoclonal antibodies can be produced by obtaining spleen cells from theanimal. (See Kohler and Milstein, Nature 256, 495-497 (1975)). In oneexample, monoclonal antibodies (mAb) of interest are prepared byimmunizing inbred mice with apoCIII, or an antigenic fragment thereof.The mice are immunized by the IP or SC route in an amount and atintervals sufficient to elicit an immune response. The mice receive aninitial immunization on day 0 and are rested for about 3 to about 30weeks. Immunized mice are given one or more booster immunizations of bythe intravenous (IV) route. Lymphocytes, from antibody positive mice areobtained by removing spleens from immunized mice by standard proceduresknown in the art. Hybridoma cells are produced by mixing the spleniclymphocytes with an appropriate fusion partner under conditions whichwill allow the formation of stable hybridomas. The antibody producingcells and fusion partner cells are fused in polyethylene glycol atconcentrations from about 30% to about 50%. Fused hybridoma cells areselected by growth in hypoxanthine, thymidine and aminopterinsupplemented Dulbecco's Modified Eagles Medium (DMEM) by proceduresknown in the art. Supernatant fluids are collected from growth positivewells and are screened for antibody production by an immunoassay such assolid phase immunoradioassay. Hybridoma cells from antibody positivewells are cloned by a technique such as the soft agar technique ofMacPherson, Soft Agar Techniques, in Tissue Culture Methods andApplications, Kruse and Paterson, Eds., Academic Press, 1973.

“Humanized antibody” refers to antibodies derived from a non-humanantibody, such as a mouse monoclonal antibody. Alternatively, humanizedantibodies can be derived from chimeric antibodies that retains orsubstantially retains the antigen-binding properties of the parental,non-human, antibody but which exhibits diminished immunogenicity ascompared to the parental antibody when administered to humans. Forexample, chimeric antibodies can comprise human and murine antibodyfragments, generally human constant and mouse variable regions. Sincehumanized antibodies are far less immunogenic in humans than thenon-human monoclonal antibodies, they are preferred for subsequenttherapeutic antibody use.

Humanized antibodies can be prepared using a variety of methods known inthe art, including but not limited to (1) grafting complementaritydetermining regions from a non-human monoclonal antibody onto a humanframework and constant region (“humanizing”), and (2) transplanting thenon-human monoclonal antibody variable domains, but “cloaking” them witha human-like surface by replacement of surface residues (“veneering”).These methods are disclosed, for example, in, e.g., Jones et al., Nature321:522-525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A.,81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988);Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun.28:489-498 (1991); Padlan, Molec. Immunol. 31(3):169-217 (1994); andKettleborough, C. A. et al., Protein Eng. 4(7):773-83 (1991).

The term antibody as used herein is intended to include antibodyfragments thereof which are selectively reactive with apoCIII, orfragments thereof. Antibodies can be fragmented using conventionaltechniques, and the fragments screened for utility in the same manner asdescribed herein for whole antibodies. For example, F(ab′)₂ fragmentscan be generated by treating antibody with pepsin. The resulting F(ab′)₂fragment can be treated to reduce disulfide bridges to produce Fab′fragments.

As used herein “selectively reactive” means that the antibodiesrecognize one or more epitope within apoCIII, but possess little or nodetectable reactivity with control proteins, such as bovine serumalbumin, under standard conditions such as those disclosed herein.

When the test compounds comprise nucleic acid sequences, such nucleicacids may be chemically synthesized or recombinantly expressed as well.Recombinant expression techniques are well known to those in the art(See, for example, Sambrook, et al., 1989, supra). The nucleic acids maybe DNA or RNA, and may be single stranded or double. Similarly, suchnucleic acids can be chemically or enzymatically synthesized by manualor automated reactions, using standard techniques in the art. Ifsynthesized chemically or by in vitro enzymatic synthesis, the nucleicacid may be purified prior to introduction into the cell. For example,the nucleic acids can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the nucleic acids may be used withno or a minimum of purification to avoid losses due to sampleprocessing.

When the test compounds comprise compounds other then polypeptides,antibodies, or nucleic acids, such compounds can be made by any of thevariety of methods in the art for conducting organic chemical synthesis.

In another aspect, the present invention provides methods for treatingpatients with type I diabetes comprising administering to the patient anamount effective of an inhibitor of apoCIII to reduce apoCIII-inducedincrease in intracellular calcium concentration in pancreatic β cells.As used herein, an “inhibitor” of apoCIII includes compounds that reducethe transcription of apoCIII DNA into RNA, compounds that reducetranslation of the apoCIII RNA into protein, and compounds that reducethe function of apoCIII protein. Such inhibiting can be completeinhibition or partial inhibition, such that the expression and/oractivity of the apoCIII is reduced, resulting in a reduced ability toincrease intracellular calcium concentration. Such inhibitors areselected from the group consisting of antibodies that bind to apoCIII;antisense oligonucleotides directed against the apoCIII protein, DNA, ormRNA; small interfering RNAs directed against the apoCIII protein, DNA,or mRNA, and any other chemical or biological compound that caninterfere with apoCIII activity. In one embodiment, the inhibitor isidentified using the compounds of the present invention. In anotherembodiment, the inhibitor is selected from the group consisting of (a)apoCIII-selective antibodies, (b) antisense nucleic acid constructsderived from the apoCIII mRNA sequence (SEQ ID NOS:1, 3, and 5) (NCBIaccession numbers X00567 (Human); J02596 (Rat); and X68359 (Macaque),respectively), and (c) small interfering RNA sequences derived from theapoCIII mRNA sequence (SEQ ID NOS:1, 3, and 5)) (NCBI accession numbersX00567 (Human); J02596 (Rat); and X68359 (Macaque), respectively).

Methods for making antibodies against apoCIII or fragments thereof aredisclosed above. Antibodies against apoCIII are commercially available(for example, from Academy BioMedical Company (Texas, USA) ChemiconInternational (California, USA); United States Biological(Massachusetts, USA), Novus Biologicals (Colorado, USA RocklandImmunochemicals (Pennsylvania, USA).

Methods for making antisense oligonucleotides and small interfering RNAsequences against the apoCIII mRNA sequence are well known to those ofskill in the art, based on the apoCIII sequences disclosed herein.Antisense oligonucleotides will be complementary to the mRNA expressedfrom the apoCIII gene, in order to bind to the mRNA to inhibittranslation.

In a preferred embodiment for using small interfering RNAs, the RNAs aredouble stranded RNAs. Methods for using such double stranded RNAs are asdescribed, for example in U.S. Pat. No. 6,506,559. For example, RNA maybe synthesized in vivo or in vitro. Endogenous RNA polymerase of thecell may mediate transcription in vivo, or cloned RNA polymerase can beused for transcription in vivo or in vitro. For transcription from atransgene in vivo or an expression construct, a regulatory region (e.g.,promoter, enhancer, silencer, splice donor and acceptor,polyadenylation) may be used to transcribe the RNA strand (or strands).The RNA strands may or may not be polyadenylated; the RNA strands may ormay not be capable of being translated into a polypeptide by a cell'stranslational apparatus. RNA may be chemically or enzymaticallysynthesized by manual or automated reactions. The RNA may be synthesizedby a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g.,T3, T7, SP6). If synthesized chemically or by in vitro enzymaticsynthesis, the RNA may be purified prior to introduction into the cell.For example, RNA can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the RNA may be used with no or aminimum of purification to avoid losses due to sample processing. TheRNA may be dried for storage or dissolved in an aqueous solution. Thesolution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

In another aspect, the present invention also provides pharmaceuticalcompositions, comprising an inhibitor of apoCIII activity and apharmaceutically acceptable carrier. In a preferred embodiment, theapoCIII inhibitor is selected from the group consisting of an antibodyreactive with apoCIII or a fragment thereof, an antisenseoligonucleotide against the apoCIII mRNA sequence, and a smallinterfering RNA sequence directed against the apoCIII mRNA sequence.

The inhibitors may be admixed with lactose, sucrose, starch powder,cellulose esters of alkanoic acids, stearic acid, talc, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine,and/or polyvinyl alcohol, and tableted or encapsulated for conventionaladministration. Alternatively, the inhibitors may be dissolved insaline, water, polyethylene glycol, propylene glycol, carboxymethylcellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseedoil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvantsand modes of administration are well known in the pharmaceutical art.The carrier or diluent may include time delay material, such as glycerylmonostearate or glyceryl distearate alone or with a wax, or othermaterials well known in the art.

The inhibitor may be made up in a solid form (including granules,powders or suppositories) or in a liquid form (e.g., solutions,suspensions, or emulsions). Inhibitor may be applied in a variety ofsolutions. Suitable solutions for use in accordance with the inventionare sterile, dissolve sufficient amounts of the antibody, and are notharmful for the proposed application. The inhibitor may be subjected toconventional pharmaceutical operations such as sterilization and/or maycontain conventional adjuvants, such as preservatives, stabilizers,wetting agents, emulsifiers, buffers etc.

As discussed below, our study shows that the sialylated forms of apoCIIIwere on average four-fold higher in sera from newly diagnosed T1Dpatients than in sera from healthy subjects.

Thus, in a further aspect, the present invention provides a method fordiagnosing Type I diabetes comprising

(a) obtaining a blood serum sample from a subject

(b) determining an amount of sialylated apoCIII in the blood serumsample; and

(c) comparing the amount with an amount of sialylated apoCIII in a bloodserum sample from a non-diabetic patient; and

(d) diagnosing those subjects with an elevated amount of sialylatedapoCIII in the blood serum sample relative to the amount of sialylatedapoCIII in a blood serum sample from a non-diabetic patient as havingtype I diabetes.

As disclosed herein, the inventor has discovered that the sialylatedforms of apoCIII predominate in type I diabetic patients, and thatanalyzing the levels of sialylated apoCIII in blood serum relative tocontrol will provide a better read out for diagnosis of type I diabetesthan analyzing apoCIII levels as a whole.

In a further embodiment, the level of mono-sialylated apoCIII in thesubject relative to control is analyzed; in another embodiment, thelevel of di-sialylated apoCIII in the subject relative to control isanalyzed; and in a further embodiment, both measurements are made.

The method does not require a specific amount of increase in sialylatedapoCIII in the blood serum sample over control, although it is preferredthat the increase is a statistically significant increase as measured bystandard statistical measurements.

The diagnostic methods of the invention can be used in combination withany other diagnostic methods known in the art, in order to increase theaccuracy of the assays.

EXAMPLES Materials and Methods

Media The basal medium used both for isolation of cells and forexperiments was a HEPES buffer (pH 7.4), containing (in mM): 125 NaCl,5.9 KCl, 1.3 CaCl₂, 1.2 MgCl₂, 25 HEPES. Bovine serum albumin was addedto the medium at a concentration of 1 mg/ml. For cell culture, RPMI 1640medium was supplemented with 100 μg/ml streptomycin, 100 IU penicillinand 10% fetal calf-, normal human- or diabetic serum.Preparation of Cells Adult mice from a local colony(3) were starvedovernight. Pancreatic islets were isolated by a collagenase techniqueand cell suspensions were prepared as previously described (4, 5). Cellswere seeded onto glass coverslips and cultured at 37° C. in a humidifiedatmosphere of 5% CO₂ in air.Preparation and Purification of Sera: Sera from T1D patients and controlsubjects were collected, identically sterile-processed and stored frozenat −20° C. until used. The sera were heat-inactivated by incubation at56° C. for 30 min. Thereafter β-cells were incubated overnight in RPMI1640 culture medium with 10% of the sera and changes in [Ca²⁺]_(i) wererecorded, subsequent to depolarization with 25 mM KCl. The five T1D serathat induced an enhanced [Ca²⁺]_(i) response were centrifuged and thesupernatant was passed through a 0.45 mm sterile filter. Samples wereloaded on Sep-Pak C₁₈ (Waters, Mass.) preconditioned with 0.1% TFA.After a wash with 0.1% TFA, proteins were eluted with 60% acetonitrilein 0.1% TFA and thereafter lyophilized. Batches of one milligram of thelyophilized sample were dissolved in 500 μl 0.1% TFA, centrifuged andinjected into a RP-HPLC with a Vydac C₁₈ (0.46×25 cm) column (GraceVydac, Hesperia, Calif.). The separation was made using a lineargradient of 20-60% acetonitrile in 0.1% TFA for 40 min at 1 ml/min.Fractions of 1 ml were collected and lyophilized.Purification of Isoforms of Apolipoprotein CIII (apoCIII) ApoCIII waspurified from human serum by adsorption to a lipid emulsion anddelipidation, followed by chromatography of the lipid-associatedproteins under denaturing conditions in guanidinium chloride and urea,respectively, as previously described (6). The apoCIII isoforms weredialyzed against ammonium bicarbonate and lyophilized before use.Measurements of [Ca²⁺]_(i) Cells, attached to coverslips, werepretreated with the different compounds as described in the results andthereafter incubated in basal medium with 2 μM fura-2AM (MolecularProbes, Eugene, Oreg.) for 30 min. The coverslips were mounted as thebottom of an open chamber and cells were perfused with medium.Fluorescence signals were recorded with a SPEX Fluorolog-2 systemconnected to an inverted Zeiss Axiovert epifluorescence microscope. Theexcitation and emission wavelengths were 340/380 and 510 nm,respectively. The results are presented as 340/380 excitation ratios,directly representative of [Ca²⁺]_(i) (7).Patch Clamp Whole-cell Ca²⁺ currents were recorded by using theperforated-patch variant of the whole-cell patch-clamp recordingtechnique to eliminate the loss of soluble cytoplasmic components.Electrodes were filled with (in mM): 76 Cs₂SO₄, 1 MgCl₂, 10 KCl, 10NaCl, and 5 HEPES (pH 7.35), as well as amphotericin B (0.24 mg/ml) topermeabilize the cell membrane and allow low-resistance electricalaccess without breaking the patch. Pancreatic β-cells were incubated inRPMI 1640 medium with apoCIII (10:g/ml) or vehicle overnight. The cellswere bathed in a solution containing (in mM): 138 NaCl, 10tetraethylammonium chloride, 10 CaCl₂, 5.6 KCl, 1.2 MgCl₂, 5 HEPES and 3D-glucose (pH 7.4). Whole-cell currents induced by voltage pulses (froma holding potential of −70 mV to several clamping potentials from −60 to50 mV in 10 mV increments, 100 ms, 0.5 Hz) were filtered at 1 kHz andrecorded. All recordings were made with an Axopatch 200 amplifier (AxonInstruments, Foster City, Calif.) at room temperature (about 22° C.).Acquisition and analysis of data were done using the software programpCLAMP6 (Axon Instruments, Foster City, Calif.).Protein Characterization Primary sequence was obtained in ABI 494C andcLC sequencers. Protein molecular weights were determined byelectrospray mass spectrometry (AutoSpec hybrid tandem massspectrometer, Micromass). For recording of positive-ion conventional-ESspectra, samples (16 pmol/ml) were introduced into the ES interface byinfusion or loop injection at a flow rate of 3 ml/min. To determine theposition of the glycosylation, the native protein was digested withtrypsin 1:10 w/w (Promega, Madison, Wis.). The resulting fragments wereseparated by HPLC using a Vydac C₈ (2.1×150 mm) and a gradient of 0-50%B in 50 min (buffer A, 5% acetonitrile/0.1% TFA; B, 80%acetonitrile/0.1% TFA). The fragments separated were applied to massanalysis.Quantification of apoCIII Sera were collected and prepared as describedabove. The relative amounts of apoCIII in T1D serum and control serum,respectively were evaluated by comparisons of the peak areacorresponding to apoCIII in the second RP-HPLC.Flow Cytometric Analysis of Cell Death RINm5F cells were cultured for 36h in the presence of 10% control serum, control serum and 40 μg/mlapoCIII or T1D serum with or without 100 or 200 μg/ml anti-apoCIII. Thewhole cell population was collected and stained with EGFP-conjugatedAnnexin V and propidium iodide (PI) (BD PharMingen) and analyzed on aFACscan using CELLQuest acquisition software (Becton Dickinson,Immunocytometry System). FACS gating, based on forward and side scatter,was used to exclude cellular debris and autofluorescence and typically10 000 cells were selected for analysis.Statistical Analysis Statistical significance was evaluated by Student'st-test and P values <0.05 were considered significant. Data areexpressed as means±SEM.Results and Discussion

ApoCIII plays a key role in the regulation of the metabolism oftriglyceride-rich lipoprotein (TRL) (8). It controls the catabolism ofTRL by inhibiting the activity of lipoproteinlipase (LPL) (9, 10),thereby inducing hypertriglyceridemia. ApoCIII also inhibits the bindingof remnant lipoproteins to catabolic receptors like the LDL receptorrelated protein (LRP) (11). When the apoCIII gene was disrupted inknock-out mice, there was a 70% reduction in triglyceride levels (12).Overexpression of human apoCIII in transgenic mice results inhypertriglyceridemia (13). ApoCIII is a 79-residue, 8.8 kDa polypeptide(14) with three known isoforms that differ in the extent ofglycosylation, CIII₀ (no sialic acid), CIII₁ (one sialic acid residue),CIII₂ (two sialic acid residues), contributing approximately 10%, 55%and 35%, respectively, of total plasma apoCIII (15). Mutagenesis of theglycosylation site and expression in stable cell lines suggest thatintracellular glycosylation is not required for the transport andsecretion functions (16). Lack of glycosylation does not affect theaffinity of apoCIII for VLDL and HDL (16). Insulin is involved in theregulation of the apoCIII gene and induces a dose-dependentdown-regulation of the transcriptional activity. Overexpression of theapoCIII gene could contribute to the hypertriglyceridemia seen in T1Dpatients (17). However, mice transgenic for the human apoCIII gene areneither insulin-resistant nor hyperinsulinemic (18). The concentrationof apoCIII has previously been found to be higher in diabetic patientsthan in normal subjects (19-27). In insulin deficient rats there was nosignificant change in apoCIII in one study (28), while others havereported an increase in the proportions of the sialylated apoCIII (29,30).

We have tested sera from seven newly diagnosed T1D patients (Table 1).Mouse pancreatic β-cells were cultured overnight with 10% sera frompatients or normal subjects. Sera from five of the patients induced asignificantly higher increase in [Ca²⁺]_(i), when cells were depolarizedwith 25 mM KCl, leading to an opening of voltage-gated L-typeCa²⁺-channels, than sera from healthy blood donors (FIG. 1). Positivesera were pooled, concentrated and fractionated by reversed phase(RP)-HPLC. When fractions were tested on isolated mouse pancreaticβ-cells, one fraction (No. 3, FIG. 2A) eluting between 52-60%acetonitrile, induced a more pronounced increase in [Ca²⁺]_(i) whencells were depolarized with high concentrations of K⁺. After furtherpurification of the component(s) in this fraction by repeated RP-HPLCruns (FIGS. 2B,D), all fractions obtained were tested for effects on[Ca²⁺]_(i) by incubation with mouse β-cells overnight. The results fromthis second purification (FIG. 2B) showed a higher activity in fraction2 (FIG. 2C). The protein that induced an increase in [Ca²⁺]_(i)indicated by the bar in FIG. 2D was determined. Sequence information wasobtained both by C-terminal and N-terminal degradations. The sequenceswere identical to those of human apoCIII for 20 N-terminal and 5C-terminal residues.

TABLE 1 Characterization of the T1D patients. Duration Age of T1DPatient Sex (years) (weeks) Medication ICA GAD IA-2 1 M 32 <1 No* PosPos Pos 2 F 32 12 No* Neg Pos Neg 3 F 31 <1 No* Pos Pos Pos 4 F 23 <1No* Pos Neg ND 5 M 19 <1 No* Neg Neg Pos 6 F 35 <1 No* Pos Pos Pos 7 F33 28 No* Pos Pos ND Gender of the patients is designated as F, female,and M, male. The presence (Pos), absence (Neg) or no data available (ND)of antibodies to islet cells (ICA), GAD and tyrosine phosphatase IA2(IA-2) are marked in the table. Healthy blood donors, all negative forICA, GAD and IA-2, served as sources of control sera. *Insulin was theonly medication administered.

We analyzed the apoCIII purified from T1D sera by mass spectrometry forsubcomponent identification. The major components had apparent masses of9423 and 9714 Da (FIG. 2E), corresponding to the mono- anddi-glycosylated forms of apoCIII (theoretical, calculated values are:CIII₀ 8764 Da, CIII₁ 9420 Da, CIII₂ 9712 Da). To determine the positionsof glycosylation, the protein was digested with trypsin and thefragments were separated by RP-HPLC. When the separated fragments wereanalyzed by mass spectrometry, seven of the eight fragments showedmasses identical to the theoretical values. The mass difference waslocalized to the C-terminal fragment, previously shown to beglycosylated (31). The absence of a non-glycosylated C-terminal fragmentindicated that the isolated apoCIII forms were glycosylated. Therelative amounts of apoCIII in T1D and control sera were evaluated bycomparisons of the peak area corresponding to apoCIII in the secondRP-HPLC (FIG. 3A). In T1D sera the levels of the sialylated isoforms ofapoCIII (apoCIII₁ and apoCIII₂) were four-fold higher than innon-diabetic sera. The non-sialylated isoform (apoCIII₀) could not bedetected.

The concentration of apoCIII has been reported to be between 6-14 mg/dlin control subjects and 9-27 mg/dl in diabetics (19, 20, 24-27). Thesevariations may to a certain extent reflect the fact that various methodshave been used for the determinations. In our experiments we have used10% T1D serum in the culture medium instead of 10% fetal calf serumnormally used, and therefore we chose to use concentrations in the range10-50 μg/ml. We have tested concentrations from 1-50 μg/ml and with 1, 3and 6 μg/ml we did not see any effects, but with the concentrations10-50 μg/ml we had responses.

Commercially available apoCIII (Sigma), which constitutes a mixture ofapoCIII₁ and apoCIII₂, was tested at a concentration of 10 μg/ml and wasshown to stimulate Ca²⁺ influx as the product isolated from T1D sera(FIG. 3B). Co-incubation of β-cells with 100 μg/ml of a polyclonalantibody against human apoCIII (Academy BioMedical Company, Houston,Tex.) blocked the activity of both the commercial apoCIII and the T1Dserum (FIGS. 3B,C). The polyclonal antibody had no activity by itself(data not shown). When testing the three isoforms of apoCIII byincubation of β-cells at a concentration of 10 μg/ml, both theglycosylated (CIII₁ and CIII₂) and the un-glycosylated isoform causedsignificantly higher increase in [Ca²⁺]_(i) than cells that had beenincubated with only the vehicle, 0.1% triflouroacetic acid (TFA) (FIG.3D). To study the effect of possible binding of apoCIII to serumlipoproteins in the culture medium, cells were incubated in basal buffercontaining no serum and 10 μg/ml apoCIII₁ for 2 and 6 h. There was asignificantly elevated increase in [Ca²⁺]_(i) upon depolarization in allthe experiments where the cells had been exposed to apoCIII₁ for 6 h,but only in one out of three experiments where the incubation time wasonly 2 h (data not shown).

There was a higher percentage of dead cells in the cell populationexposed to T1D serum. This effect was prevented by the addition ofanti-apoCIII (FIG. 3E). Furthermore, the addition of pure apoCIII toculture medium with control serum resulted in an increased cell death.

To elucidate the molecular mechanism underlying the stimulatory effectof apoCIII on [Ca²⁺]_(i), the activity of voltage-gated Ca²⁺-channelswas analysed in β-cells incubated with 10 μg/ml apoCIII. ApoCIII-treatedcells displayed larger Ca²⁺-channel currents than control cells duringdepolarizations in the range -10 to 10 mV, from a holding potential of−70 mV (FIGS. 4A,B). These data demonstrate that apoCIII modulated theactivity of the voltage-gated L-type Ca²⁺-channel and that the effectoccurred in the range of physiological depolarizations. So farimmunoblot experiments have not revealed a direct interaction of apoCIIIwith the Ca²⁺-channel (data not shown). Future experiment will clarifyto what extent this reflects imperfectness in the immunoprecipitationprotocol or the actual true situation.

Our study shows that the sialylated forms of apoCIII were on averagefour-fold higher in sera from newly diagnosed T1D patients than in serafrom healthy subjects. ApoCIII induced both an increase in [Ca²⁺]_(i)and β-cell death. The molecular mechanism underlying the stimulatoryeffect of apoCIII on [Ca²⁺]_(i) reflected an activation of thevoltage-gated L-type Ca²⁺-channel. Addition of an antibody againstapoCIII blocked the effects of both T1D serum and apoCIII on [Ca²⁺]_(i)as well as on β-cell death. This suggests that the Ca²⁺ dependentcytotoxic effect of T1D serum on the pancreatic β-cell is mediated byapoCIII.

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1. A method for limiting pancreatic β cell apoptosis comprisingadministering to a patient with type I diabetes an amount effective ofan inhibitor of human apoCIII to reduce apoCIII-induced increase inintracellular calcium concentration in pancreatic β cells, wherein theinhibitor is selected from the group consisting of an apoCIII-selectiveantibody, an antisense oligonucleotide directed against the apoCIIImRNA, and a small interfering RNA directed against the apoCIII mRNA. 2.The method of claim 1 wherein the patient has an increased blood serumlevel of human apoCIII.
 3. The method of claim 1, wherein the patienthas an increased blood serum level of sialylated human apoCIII.
 4. Themethod of claim 1, wherein the inhibitor is an apoCIII-selectiveantibody.
 5. The method of claim 1, wherein the inhibitor is anantisense oligonucleotide directed against the apoCIII mRNA.
 6. Themethod of claim 1, wherein the inhibitor is a small interfering RNAdirected against the apoCIII mRNA.